“Biochips” or arrays (also known as microarrays) of binding agents, such as oligonucleotides, cDNA and peptides, and the like have become an increasingly important tool in the biotechnology industry and related fields. These binding agent arrays, in which a plurality of binding agents are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like.
In array-based assays in which an array of binding agents is employed, the array surface is reacted with one or more analytes, such as polynucleotide analytes, receptor proteins or antiligand molecules, under conditions that promote specific, high-affinity binding of the analyte molecules to one or more of the array members. Typically, the goal is to identify one or more position-addressable members of the library array which bind to the analyte, as a method of screening for array compounds which bind the analyte. Typically, the analyte is labeled with a detectable reporter such as a fluorescent agent, which in effect can fluorescently label the one or more array regions where analyte binding to the array has occurred.
Once the binding of the analyte to one or more array members has occurred, the arrays are read, usually by optical means, where a variety of optical scanning devices have been proposed for reading such arrays (see for example U.S. Pat. Nos. 5,324,633 and 5,585,639, the disclosures of which are herein incorporated by reference). The optical means included in these array scanning devices typically includes a light source, e.g., a laser, photodiode or the like, for transmitting light onto the array and a detector, e.g., a photomultiplier or the like, for detecting a parameter of the transmitted light, e.g., light absorption, fluorescence, etc. Typically, a microprocessor working under the control of a software program is associated with the device, which microprocessor processes the information received by the detector, for example it performs calculations, comparisons and the like.
As will be apparent, it is imperative that the optical means in these scanners performs consistently over time. In other words, it is important that the light source and detector accurately and precisely detect fluorescently labeled regions on an array surface and that such detection is consistent amongst scanners. Thus, it would be advantageous if array optical scanners could be periodically calibrated to achieve and maintain such consistency, precision, accuracy, etc., and also to ensure that variations between optical scanners are minimized, i.e., each optical scanner produces substantially the same results as any other scanner. More precisely, it would be advantageous if the optical components of the scanner, e.g., the light source and/or light detector and certain other optical components of the system including the scanning lens(es), optical stage and the scanner mirror(s), were periodically checked and, if necessary, re-adjusted.
Typically, the optical means of a scanner are calibrated during manufacture. Methods and devices are known for calibrating light sources (see for example U.S. Pat. Nos. 5,464,960 and 5,772,656). However, few are known for calibrating optical components, such as an optical detector for example, after manufacture. Thus, optical detectors and various other optical components are usually not calibrated periodically, due to the lack of an easy, precise and inexpensive end-user calibration tool. However, one method that has been developed to calibrate optical detectors of scanners after manufacture uses a substrate having a pattern of reflective metal, typically chromium, thereon. The method relies solely on the reflection of light from the chromium pattern, where such reflective light measurements are used to calibrate the optical detector of the scanner. One disadvantage with this system is that although it is capable of calibrating the scanner's positioning mechanism with high precision, it lacks the capability to detect power changes in the laser or reduced responses in the detector. Another disadvantage associated with this method is that it is wavelength insensitive. In other words, using reflectance to calibrate the optical detector of an optical scanner that is intended to scan fluorescently labeled probes, does not evaluate the optical detector of the scanner in regards to the wavelength of light that will ultimately be emitted, e.g., fluorescence, when an actual array is scanned by the optical scanner. Another significant disadvantage is that the chromium tool does not include a means to subtract background signal from the reflectance data, aside from the dark current typical of all detectors, and thus the reflectance data may be over or under estimated which may then skew the calibration of the optics.
To be a suitable reference, the reference should provide a stable output, preferably over a significant period of time, should have minimal local and global nonuniformities, should not be substantially degraded by the optics of the scanner, should also produce a stable output at the frequency or wavelength range of interest, i.e., corresponding to a wavelength/frequency emitted from an actual scanned array (e.g., emitted from fluorescently labeled probes) and a means to measure and subtract background signal. As such, there is continued interest in the development of new devices for optical scanner calibration devices and methods of using the same. Of particular interest would be the development of an array optical scanner calibration device, and methods of use thereof, that produces a stable output at the frequency or wavelength of interest, has minimal local and global nonuniformities, and is easy to manufacture and use.
Relevant Literature
U.S. Patent documents of interest include U.S. Pat. Nos. 4,868,105; 5,124,246; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,445,934; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,556,752; 5,561,071; 5,563,034; 5,585,639; 5,599,695; 5,624,711; 5,631,734; 5,639,603; 5,658,734; 5,681,702; and 5,981,956. Other documents of interest include WO 93/17126; WO 95/11995; WO 95/35505; WO 97/14706 and WO 98/30575; WO 98/24933; EP 742287; EP 799897; WO 01/59503; Chen Y., et al., Journal of Biomedical Optics (1997) 2:364–374; and DeRisi J. L. et al. (1997) Science 278:680–686.